Jang, Yeon MD; Kim, Eun S. MD; Park, Soo S. MD; Lee, Jaemin MD; Moon, Dong E. MD
It has been speculated that there are similarities between pathophysiological and biochemical mechanisms observed in convulsions and in neuropathic pain, and this has led to the use of anticonvulsants for the treatment of neuropathic pain (1). The anticonvulsant carbamazepine (CBZ) is effective in managing neuropathic pain (2). Oxcarbazepine (OCBZ), a 10-keto analog of CBZ, has been reported to be equally effective as CBZ in the treatment of trigeminal neuralgia (3). OCBZ induces fewer drug interactions and has fewer adverse effects than CBZ3. In one clinical report, it was suggested that OCBZ is useful against various types of neuropathic pain, such as painful diabetic neuropathy and complex regional pain syndrome (4). However, there have been few controlled studies on the effects and adverse reactions of OCBZ in animal pain models.
In this study, we examined the antiallodynic effect of OCBZ by observing withdrawal responses to mechanical and cold stimuli in a rat model of neuropathic pain to determine whether OCBZ may offer a new treatment modality for neuropathic pain diseases.
All experimental procedures were approved by our institutional animal investigation committee. Male Sprague-Dawley rats weighing 100–150 g were used. The rats were housed in groups of 3–4 in plastic cages with soft bedding and maintained on a 12:12-h light-dark cycle. Experimental rats were adjusted to the environment for at least 5 days before conducting this experiment. The Kim and Chung (5) method was used to produce the neuropathic pain model by ligating the left L5 and L6 spinal nerves. After surgery, the rats were allowed to recover for 7 days before behavioral tests. Rats showing a foot withdrawal response to von Frey filaments (18011 Semmes-Weinstein filament, Stoelting, CO) with a bending force of 35.6 mN or less were considered neuropathic and were then used in the tests. Rats that exhibited motor deficiency (such as paw dragging or limping) or failure to exhibit subsequent mechanical allodynia were excluded from further testing.
Sixty neuropathic rats were randomly divided into six groups (n = 10 in each group) before the intraperitoneal administration of drugs. In these rats, the intraperitoneal injections were performed under enflurane (2.0 vol%) anesthesia 15 min before behavior tests. Because the vehicle used to dissolve the drug may induce sedation and influence pain responses, we included both a vehicle-control group and a saline-control group. The saline-control group received only normal saline, and the vehicle-control group received 30% polyethylene glycol 400 (PEG 400). Experimental groups were divided into four groups (OX 10, OX 20, OX 30, and OX 50), and different doses of OCBZ were administered to each group: 10 mg/kg, 20 mg/kg, 30 mg/kg, and 50 mg/kg, respectively. OCBZ (Trileptal®, Novartis, Switzerland™) was dissolved into 30% PEG 400 (Sigma Chemical, Fluka, Switzerland) for injection. Intraperitoneal injections were uniformly given at a volume of 10 mL/kg.
All behavioral tests were conducted at fixed times (1:00–5:00 pm) in a quiet room by the same person who was kept unapprised of both the injected solution and the dose used. After intraperitoneal injection, the rats were placed on a metal mesh covered with a plastic dome (8 × 8 × 18 cm) for the assessment of mechanical and cold allodynia. Rats were allowed to adapt for at least 20 min before the commencement of testing. Mechanical and cold allodynia were assessed both before intraperitoneal injection and at 15, 30, 60, 90, 120, 150, and 180 min after. All behavioral studies (von Frey, acetone, and rotarod) were started from the postoperative first week and performed for 3–4 wk. Each rat was tested for mechanical and cold allodynia three times, one test a week. A motor coordination test (rotarod) was performed three times alternatively with the same intervals. To avoid the residual drug effects, each experiment was performed at least 3 days after the preceding test (von Frey, acetone, or rotarod). Allodynia was measured during each new experiment, and rats with an allodynia level of 35.6 mN or more were excluded from further testing. The dosage regimen and observation times were based on the preliminary experimental results and previous experience. At the end of the experiments, rats were killed by anesthetic overdose.
The von Frey filament, manufactured to apply 35.6 mN of pressure, was used for measurements. Mechanical stimuli were given 10 times through the wire mesh to the plantar surface (third metatarsal bone area) of the left hindpaw for which the nerve root had been ligated. Mechanical stimuli were administered at intervals of 3–4 s. The occurrence of foot withdrawal in each of these 10 trials was expressed as a percent response frequency ([number of paw withdrawals/number of trials] × 100). Avoidance responses such as lifting, shaking, or licking the paw and running away were regarded as positive responses.
The thresholds for mechanical allodynia were measured with a series of 12 von Frey filaments ranging from 1.6 mN to 278.4 mN. The third metatarsal bone area of the left hindpaw was stimulated three times with von Frey filaments at 30-s intervals. The minimal pressure level (milli-Newtons [mN]) that initiated a response was recorded as a median value. The testing was performed by the up-down method, and if the strongest hair did not elicit a response, threshold was recorded as 743.7 mN.
Cold allodynia was measured as the number of foot withdrawal responses after application of cold stimuli to the plantar surface of the paw6. A drop of 100% acetone was gently applied to the heel of the rat with a syringe connected to a thin polyethylene tube. A brisk foot withdrawal response after the spread of acetone over the plantar surface of the paw was considered a sign of cold allodynia. The testing was repeated 5 times with an interval of approximately 3–5 min between each test. The same standard was applied to positive withdrawals as for mechanical stimuli. The response frequency to acetone was expressed as a percent response frequency ([number of paw withdrawals/number of trials] × 100).
Locomotor function changes in the neuropathic rats were evaluated by rotarod testing (Acceler rota-rod for rats 7750; Ugo Basile, Comerio-Varese, Italy). Neuropathic rats were acclimated to revolving drums and habituated to handling to ameliorate stress during testing. Before the actual day of drug testing, rats were given three training trials on revolving drums (10–15 rpm) for 2 days. Rats able to remain on the revolving drum for a minimum of 150 s were selected for drug testing. The mean of 3 training runs served as a control performance time. Rotarod performance time was measured at 20, 60, 90, 120, and 150 min after intraperitoneal injection. Each test was performed three times at 5-min intervals, and the mean values were compared.
The results were expressed as the mean ± sem or median value. Statistical analysis was performed with Sigma-Stat (version 2.03; SPSS Inc, Chicago, IL). Percent response frequency and rotarod performance time were assessed using repeated-measure analysis of variance, followed by post hoc Dunnett tests for multiple comparisons. Withdrawal thresholds were compared using Kruskal-Wallis analyses of variance on rank. A P value of < 0.05 was considered significant.
Neuropathic pain behavior developed within 2–3 days after spinal nerve ligation in 80% of experimental rats. These rats looked healthy, without any complications such as paralysis or limping. During the 4-wk test period, mechanical and cold allodynia were consistently maintained.
In all rats, withdrawal frequencies were 95% or more at the preadministration time, and there were no significant differences among the six groups. Fifteen minutes after drug administration, all rats showed minimal withdrawal frequency, and then withdrawal frequencies increased gradually. In the 2 control groups (saline and vehicle), there were no significant differences in withdrawal frequency compared with preadministration values except for the one measured at 15 min. In the OCBZ-treated groups, withdrawal frequency after drug administration decreased in a dose-dependent manner (P < 0.05; Fig. 1).
Before drug administration, there were no significant differences among all groups in median values of withdrawal thresholds. OCBZ treatment increased withdrawal thresholds at doses in the range of 10–50 mg/kg (P < 0.05). The duration of this effect was dose dependent, and in the 50-mg/kg dose, the increased withdrawal thresholds persisted for 2 h (Fig. 2).
Before drug administration, all groups showed withdrawal frequencies of 90% or more after 100% acetone stimuli, and there were no significant differences among all six groups. OCBZ treatments reduced response frequency to acetone in a dose-dependant manner (P < 0.05; Fig. 3).
We observed no significant change in rotarod performance time at OCBZ dosages between 10–30 mg/kg. By contrast, at a dose of 50 mg/kg, OCBZ significantly decreased locomotor function from 20 to 120 min after drug administration. At this dose, after drug injection and before performing rotarod tests, 6 of 10 rats lay face down on the plastic experimental stand and did not respond to stimuli, which was evidence of severe sedation. Among these rats, three hung onto the rotarod for a short time and then fell off the rotarod because of sedation and locomotor dysfunction. Rats exhibiting sedation and locomotor dysfunction progressively recovered, and normal posture and response were completely regained within 90 min. In the OCBZ 30-mg/kg treated group (n = 10), 5 rats showed slight sedation after intraperitoneal injection and before commencing rotarod testing. Mean rotarod performance time was decreased to 133.3 ± 5.6 s, but this was not statistically significant, considering the 150-s cutoff time (Fig. 4).
The present study reveals that intraperitoneally administered OCBZ had a dose-dependent antiallodynic effect in a rat neuropathic model that did not produce sedation, suggesting that OCBZ treatment may provide effective therapy for neuropathic pain diseases. Considering that neuropathic pain diseases respond poorly to traditional analgesics, such as opioids or nonsteroidal antiinflammatory drugs, offering another treatment modality for neuropathic pain diseases is important.
The pain model used in this study was devised by Kim and Chung (5). In this model, ligation of L5 and L6 spinal nerves produces behavioral signs corresponding to major components of human neuropathic pain (continuing pain, mechanical and cold allodynia, and heat hyperalgesia). Features of this pain model are similar to those of clinical neuropathic pain (5,7,8). Mechanical allodynia in this pain model begins a day after nerve ligation, reaches maximum response in 2 weeks, and continues for up to 10 weeks (5,7). In our study, behavioral tests began one week after surgery, which was the active period of pain response, and were conducted repeatedly for three to four weeks. Before every drug administration, we measured the level of mechanical and cold allodynia and found that allodynia-induced pain responses continued throughout the entire experiment.
In this experiment, two methods were used to measure mechanical allodynia: observation of withdrawal thresholds on the stimuli elicited by a series of von Frey filaments and withdrawal response frequency to 35.6 mN of von Frey filaments. Observing withdrawal thresholds is the generalized method for measuring mechanical allodynia. The withdrawal frequency method was introduced by Kim and Chung (5) and has the advantage of easy quantification of responses to mechanical stimulations produced by different pressures. In the present experiment, pain responses were the lowest at 15 and 30 minutes after drug administration in all groups and then gradually returned to preadministration levels. This period may be the maximum effective period for intraperitoneally administered OCBZ. However, even in the normal saline-control and vehicle-control groups, pain responses were decreased 15 minutes after drug administration compared to preadministration values (Fig. 1), and it is possible that these values reflect residual effects from the inhaled anesthetics used during drug administration.
As mentioned earlier, the results of this study suggest that OCBZ might be used as an effective therapeutic drug for various neuropathic pain conditions that are accompanied by allodynia and hyperalgesia. Recently, Kiguchi et al. (9) reported that OCBZ increased thermal and mechanical nociceptive thresholds in streptozotocin-induced diabetic rat and mice models. These results are consistent with those of our study. In contrast, Fox et al. (10), in their neuropathic pain model of rats, reported that OCBZ administration did not affect mechanical hyperalgesia. The reason for these discrepancies between their results and ours is not clear. It may be related to the difference in pain models (spinal nerve ligation versus partial ligation of sciatic nerve), the route of drug administration (intraperitoneal versus oral), or the types of nociceptive stimulation used in the studies. The oral administration method used by Fox et al. (10) has disadvantages, such as difficulty in dosage control, inconsistent absorbance, and inadequate therapeutic blood concentration because of the reduced bioavailability caused by first pass effects. To eliminate differences depending on drug administration methods it may be required to measure the blood concentration of this drug. However, in their neuropathic pain model of guinea pigs, Fox et al. (10) reported that OCBZ produced up to 90% reversal of mechanical hyperalgesia. This result suggests that experiments performed in the same condition using the same pain models can produce different results depending on the subjects. Therefore, we should consider species factors in experimental drug studies.
Common side effects of anticonvulsants are sedation and motor dysfunction. Half of patients who take CBZ report experiencing somnolence, dizziness, and gait disturbance1. The extent and frequency of symptoms are less for OCBZ than for CBZ, but an increased dosage may cause fatigue, headache, dizziness, and gait disturbance (11). In animal behavioral tests, severe sedation after drug administration may reduce response to stimulations and mask the antiallodynic effect. Therefore, we tested rotarod performance to examine drug-induced adverse effects, such as sedation or locomotor dysfunction. Experimental rats that adjusted to the rotarod did not show significant reduction in rotarod performance when 10–30 mg/kg of OCBZ was administered. Thus, we conclude that the antiallodynic effect produced by 10–30 mg/kg of OCBZ was not caused by drug-induced sedation.
In the study of oil-soluble drugs such as OCBZ or CBZ, one of the most important factors determining the outcome is the effect of the vehicle used to dissolve the drugs. Loscher et al. (12) reported that solvents such as glycofurol, if given at 10% or larger concentration, increased convulsion thresholds and could greatly affect results. On the contrary, PEG 400 was not reported to affect electrical or chemical convulsion thresholds in concentrations of up to 30%. Loscher et al. (12) recommended that the dose should not exceed 1 mL/100 g of body weight when using PEG 400 as a solvent, and its concentration should be 30% or less. In the present study, PEG 400 was diluted with normal saline to produce a 30% solution. The administered dose of the solvent was adjusted to 1 mL/100 g of body weight. With this concentration and dose, there were no significant differences between the vehicle group and saline group with respect to withdrawal response or locomotor function. However, upon increasing the dose of OCBZ in 10-mg units, the maximum dose possible to dissolve was 50 mg per 10 mL of solvent with this concentration of the vehicle. A suspension formed at more than this concentration. Such properties of drugs and solvents should be considered in follow-up clinical studies.
The mechanism of action for OCBZ has been mainly addressed with regard to anticonvulsive effects (13–16), and the analgesic mechanisms of OCBZ have not been systematically researched. However, in some animal studies on chronic pain, it was reported that the effects of CBZ on chronic pain were associated with the N-methyl-d-aspartic acid (NMDA) receptor (17) and antiinflammatory effects (18). Fujiwara et al. (19) proved in their in vitro study with white rat brain that CBZ and OCBZ had high affinity for the adenosine A1 receptor. The study of Mashimoto et al. (20) describes that the antinociceptive effects of CBZ are related to the revitalization of the opioid system through the activation of the adenosine A1 receptor. Recently, Ichikawa et al. (21) and Kiguchi et al. (22) suggested the possible effectiveness of OCBZ in the treatment of pain syndrome in their electrophysiologic study using a cat's peripheral nerves. In their study, IV administration and intraneural microinjection of OCBZ suppressed high-frequency nerve firing, a common phenomenon in cases of neuropathic pain and convulsion, and also caused dose-dependent inhibition of C-fiber-mediated neurotransmissions. It has been accepted that the analgesic mechanisms of OCBZ and CBZ mainly involve the inhibition of voltage-dependent sodium channels (23). In this behavioral study, the antiallodynic effect of OCBZ could be ascertained through the results, but the clear mechanism of OCBZ in attenuating allodynia was not revealed. However, considering past research on mechanisms related to mechanical and cold allodynia, it could be hypothesized that OCBZ directly or indirectly affects sensitization of C-nociceptors, neuroplastic changes in myelinated A fibers, and central sensitization by means of activation of NMDA receptors. And, like CBZ, the analgesic effect of OCBZ may result from the activation of the central purinergic (adenosine A1 receptor) system and opioid system. To find the sites of drug action and precise mechanisms of action, further studies incorporating various methods, such as intrathecal or intraventricular drug administration and concomitant administration of NMDA antagonists or adenosine receptor antagonists, would be required.
In conclusion, we have demonstrated that intraperitoneally administered OCBZ had a dose-dependent antiallodynic effect in our neuropathic rat model. Proper monitoring for adverse effects, such as sedation and motor dysfunction, would be required for large doses. Our results suggest that OCBZ treatment may be one treatment modality for a broad range of neuropathic pain diseases that are accompanied by allodynia or hyperalgesia.
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